Given its central role in asthmatic airflow obstruction, airway smooth muscle (ASM) contraction has long received attention as a therapeutic target, with strategies directed at relaxing ASM or ablating ASM altogether. Here, we propose an entirely novel approach to relieving airflow obstruction in asthma. Our strategy is to impair the ability of contracted ASM to remain shortened after contraction has occurred. While ASM that has contracted against a steady load can remain shortened indefinitely, ASM contracting against a fluctuating load first shortens then relengthens;such force fluctuation-induced relengthening (FFIR) can be quite substantial and can be further enhanced pharmacologically. In the intact lung, ASM is constantly exposed to force fluctuations imposed by tidal breathing. The key premise of this application is that drugs targeted specifically to exaggerate breathing-induced FFIR should release ASM's squeeze on the airway lumen and thus relieve bronchoconstriction. Our objective is to identify and evaluate compounds that target this extremely potent but largely unstudied bronchodilatory pathway. Preliminary studies disclose disruption of actin-myosin-actin connectivity (AMAC) as an attractive strategy for enhancing FFIR. In isolated ASM, interventions that reduce the polymerization of actin or myosin filaments both reduce AMAC and promote FFIR. In a staged screening design, we will use large scale high throughput primary assays to identify novel or already-in-human compounds that: 1) inhibit polymerization of myosin into thick myofilaments;and/or 2) reduce force generation by cultured human ASM. A novel low throughput assay will then be used to test which of these compounds potentiates FFIR or inhibits bronchoconstriction in intact airways within precision- cut thin slices of human lungs. Finally, we will evaluate mechanisms of drug action by quantifying myofila}} ment lengths in flash frozen control and drug-treated intact human ASM using 3D reconstructions of myofila}} ment ultrastructure from EM tomograms, and by assessing potential alternative mechanisms of drug action, including alteration of intracellular calcium mobilization, MLC20 phosphorylation, and/or HSP27 phosphoryl}} ation. Our results should identify novel drugs for asthma that potentiate FFIR or inhibit bronchoconstriction. RELEVANCE (See instructions): The objective of this project is to identify drugs that could exert a novel therapeutic effect in asthma - by promoting the rapid reversal of bronchoconstriction. This approach is fully complementary to current therapeutic approaches aimed at suppressing airway inflammation or relaxing airway muscle and so, if developed successfully, should add an entirely new therapeutic strategy to existing asthma therapies.